DOC2B promotes insulin sensitivity in mice via a novel KLC1-dependent mechanism in skeletal muscle

Abstract

Aims/hypothesis Skeletal muscle accounts for >80% of insulin-stimulated glucose uptake; dysfunction of this process underlies insulin resistance and type 2 diabetes. Insulin sensitivity is impaired in mice deficient in the double C2 domain β (DOC2B) protein, while whole-body overexpression of DOC2B enhances insulin sensitivity. Whether insulin sensitivity in the skeletal muscle is affected directly by DOC2B or is secondary to an effect on other tissues is unknown; the underlying molecular mechanisms also remain unclear. Methods Human skeletal muscle samples from non-diabetic or type 2 diabetic donors were evaluated for loss of DOC2B during diabetes development. For in vivo analysis, new doxycycline-inducible skeletal-muscle-specific Doc2b-overexpressing mice fed standard or high-fat diets were evaluated for insulin and glucose tolerance, and insulin-stimulated GLUT4 accumulation at the plasma membrane (PM). For in vitro analyses, a DOC2B-overexpressing L6-GLUT4-myc myoblast/myotube culture system was coupled with an insulin resistance paradigm. Biochemical and molecular biology methods such as site-directed mutagenesis, coimmunoprecipitation and mass spectrometry were used to identify the molecular mechanisms linking insulin stimulation to DOC2B. Results We identified loss of DOC2B (55% reduction in RNA and 40% reduction in protein) in the skeletal muscle of human donors with type 2 diabetes. Furthermore, inducible enrichment of DOC2B in skeletal muscle of transgenic mice enhanced whole-body glucose tolerance (AUC decreased by 25% for female mice) and peripheral insulin sensitivity (area over the curve increased by 20% and 26% for female and male mice, respectively) in vivo, underpinned by enhanced insulin-stimulated GLUT4 accumulation at the PM. Moreover, DOC2B enrichment in skeletal muscle protected mice from high-fat-diet-induced peripheral insulin resistance, despite the persistence of obesity. In L6-GLUT4-myc myoblasts, DOC2B enrichment was sufficient to preserve normal insulin-stimulated GLUT4 accumulation at the PM in cells exposed to diabetogenic stimuli. We further identified that DOC2B is phosphorylated on insulin stimulation, enhancing its interaction with a microtubule motor protein, kinesin light chain 1 (KLC1). Mutation of Y301 in DOC2B blocked the insulin-stimulated phosphorylation of DOC2B and interaction with KLC1, and it blunted the ability of DOC2B to enhance insulin-stimulated GLUT4 accumulation at the PM. Conclusions/interpretation These results suggest that DOC2B collaborates with KLC1 to regulate insulin-stimulated GLUT4 accumulation at the PM and regulates insulin sensitivity. Our observation provides a basis for pursuing DOC2B as a novel drug target in the muscle to prevent/treat type 2 diabetes. Karla E. Merz and Arianne Aslamy contributed equally to this work.Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00125-019-4824-2) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Synaptotagmin-1 and Doc2b Exhibit Distinct Membrane-Remodeling Mechanisms

“…In neurons, Syt1 and Doc2b each contribute to synaptic vesicle secretion but exhibit very distinct kinetics: Syt1 triggers fast synchronous neurotransmission evoked by action potentials and the subsequent opening of voltage-gated Ca 2þ channels (41), whereas Doc2b contributes to the socalled spontaneous release events that occur in the absence of action potentials (34). In addition, Doc2b is also implicated in the secretion of chromaffin granules (37,42) and insulin (43) and the externalization of glucose transporters (44,45).…”

Key circular RNAs identified in male osteoporosis patients by whole transcriptome sequencing

“…Full-size DOI: 10.7717/peerj.11420/fig- 5 Qu, Chu & Wang (2017) demonstrated that miR-195-5p suppresses osteosarcoma cell proliferation and invasion by suppressing naked cuticle homolog 1. Zhang et al (2019) discovered that DOC2B promoted insulin sensitivity in mice via a novel KLC1-dependent mechanism in skeletal muscle. Importantly, we found that circRNA hsa_circ_0042409 and KLC1 mRNA were significantly increased while hsa-miR-195-5p was significantly decreased in male osteoporosis patients by whole transcriptome sequencing and further validated by qPCR.…”

Conventional and Unconventional Mechanisms by which Exocytosis Proteins Oversee β-cell Function and Protection

“…In line with this, both STX4 and DOC2b is known to associate with microtubule-associated Tctex-1 type proteins, reinforcing the regulatory contribution of both of these exocytosis proteins in cytoskeletal remodeling [ 84 , 91 , 92 ]. Recently, DOC2b was shown to undergo tyrosine-phosphorylation at tyrosine 301, within its functionally indispensable C2B domain following insulin stimulation in the skeletal muscle, resulting in its association with microtubule protein kinesin light chain 1 (KLC1) [ 93 ]. KLC1 is a cytoskeletal motor protein with a proposed role in protein/vesicle translocation.…”

“…KLC1 is a cytoskeletal motor protein with a proposed role in protein/vesicle translocation. The association of DOC2b-KLC1 is critical for its stimulatory role in glucose uptake in the skeletal muscle [ 93 ]. However, the precise contribution of DOC2b in cytoskeletal remodeling in the β-cell during insulin exocytosis is not known and calls for future investigation ( Figure 4 ).…”

“…DOC2b enrichment protects β-cells from cytokine-induced apoptosis and ameliorates ER stress, and a truncated portion of the DOC2b protein containing only the two C2 domains is needed to confer this resilience [ 78 ]. Post-translational modification of tyrosine 301 within the C2B domain of DOC2b was found to confer resilience to stressed skeletal muscle cells [ 93 ], and may function similarly in β-cells. Examination of nonconventional binding interactions, as well as their susceptibilities to stress-induced post-transcriptional/translational modifications, will provide insight into how the SNARE complexes function and how to stabilize them to confer β-cell resilience against diabetogenic stress.…”